Objectives Borrelia burgdorferi, the spirochete that causes Lyme disease, expresses a surface lipoprotein, VlsE, which undergoes antigenic variation (Zhang et al., Cell. 89:275-285, Infect. Immun. 66:3698-3704). Variation takes place via a unidirectional recombination mechanism whereby vlsE exchanges portions of a central ?cassette? segment of the molecule with 15 similar cassettes located upstream from the vlsE locus (Ibid.). The N- and C- terminal domains of VlsE, each roughly 1/3 of the molecule?s length, as well as several regions within the cassette, remain invariant (Ibid.). Nothing is known about the functional or antigenic properties of these invariant regions. We set out to determine these properties, as such invariant regions could be potential diagnostic reagents or vaccinogens. Results We cloned and sequenced a cassette segment of the vls locus of the IP90 strain of Borrelia garinii, expressed it in Escherichia coli and purified the corresponding polypeptide (P7-1). We an alyzed the antigenicity of the invariant regions (IRs) of P7-1 using standard algorithms, and determined the percent identity of their amino acid sequences with the corresponding IRs of published VlsE cassettes of B. burgdorferi ss strains B31 and 297. One IR, namely IR6, showed the highest level of identity among Borrelia species and strains, and exhibited a stretch of 7 amino acids which was predicted to be antigenic by the Hopp-Woods algorithm. We synthesized a peptide, C6, which encompassed the aa sequence of IR6, and assessed its antigenicity in B. burgdorferi-infected mice, monkeys and humans. We also performed antigenic competition experiments, to determine what proportion of B. burgdorferi-infected mouse, monkey and human antibody responses to P7-1 was directed to C6. External exposure of IR6 on the VlsE molecule was determined by immunoprecipitation of detergent-solubilized, native VlsE of IP90 with rabbit antisera raised against C6, and exposure of IR6 on the spirochetal surf ace was assessed by immunofluorescence and antibody-dependent, complement-mediated killing assays with the anti-C6 antibody. The results indicate that IR6 is exposed on the VlsE ?surface? but not on the surface of the spirochete; it is immunodominant and may thus contribute to focus the antibody response onto a conserved section of VlsE. Future directions Extend this antigenic analysis to the additional 5 invariant regions of VlsE. FUNDING Grant from SmithKline Beecham Biologicals and Base grant. PUBLICATIONS None